Myofibroblasts are found in abundance at sites of wound healing and fibrosis. The origin and genesis of these cells, and their roles in the pathogenesis of fibrotic lesions, is unclear. There is evidence that these cells may be responsible for wound contraction as well as contributing to increased contractility of the lung in pulmonary fibrosis, in view of their increased content of microfilaments and alpha-smooth muscle actin. Although there is suggestive evidence that these cells may be the source of much of the collagen laid down in fibrotic lungs, direct evidence for such a role is lacking. The long term objective of this application is to elucidate the genesis and role of the myofibroblast in pulmonary fibrosis. Preliminary evidence generated for this application provides direct evidence that most of the collagen expressing cells in a rat model of bleomycin-induced pulmonary fibrosis are positive for alpha- smooth muscle actin. The preliminary data also indicate that the peak of actin expression approximates that for the peak of type I collagen mRNA expression. This peak of actin and collagen expression is accompanied by peak increases in lung expression of tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta-1) and monocyte chemotactic protein-1 (MCP-1). Previous in vitro studies also indicate that certain factors, such as extracellular matrix components and cytokines, can modulate alpha-smooth muscle actin expression in isolated fibroblasts and other cell lines. Based on these studies and preliminary data, the following hypothesis is proposed. Increased lung cytokine production in pulmonary fibrosis results in upregulation of alpha-smooth muscle actin expression in fibroblasts, giving rise to the myofibroblast phenotype. These myofibroblasts then are responsible for the increased collagen production characteristic of lung fibrosis. Additionally, it is hypothesized that these myofibroblasts have an increased capacity to elaborate more cytokines which could participate in amplifying the fibrotic process by paracrine and autocrine mechanisms. This in turn may be associated with increased contractility of the fibrotic lung. To test this hypothesis, five major specific aims are proposed: 1) determine the kinetics of the appearance of myofibroblasts in bleomycin- induced pulmonary fibrosis and correlate this with that for type I collagen expression; 2) assess expression of TGF-beta-1 and MCP-1 by myofibroblasts; 3) evaluate the mechanism of emergence of the myofibroblast phenotype in vivo and in vitro; 4) determine functional parameters (collagen synthesis, cytokine production and collagen gel contraction) of myofibroblasts of relevance to pulmonary fibrosis; and 5) assess the importance of alpha-smooth muscle actin expression to the functions determined in aim 4. The aims will be approached using a combination of in situ hybridization, Northern analysis and immunohistochemistry to assess cytokine, actin and collagen gene expression, as well as for identification of cell type. An antisense strategy will be employed in aim 5. This combined in vivo and in vitro approach should provide new insights as to the genesis of the myofibroblast and its role in pulmonary fibrosis.